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De novo mutations arise spontaneously in the egg or sperm and are not inherited from either parent.

Detecting mutations in IVF embryos

Pre-implantation genetic diagnosis (PGD) is used in fertility clinics to detect abnormalities before they are passed on by parents to their in vitro fertilized (IVF) embryos. However, it has not been possible to scan an embryo's genome to detect mutations — until now perhaps.

In a study published in Genome Research, scientists have developed a whole-genome sequencing approach using 5 to 10 cells from IVF embryos to detect potential disease-causing mutations.

Researchers from Complete Genomics, Reprogenetics, and the NYU Fertility Center have now sequenced three biopsies from two IVF embryos in order to detect de novo mutations — those that arise spontaneously in the egg or sperm and are not inherited from either parent.

De novo mutations are thought to account for a large fraction of severe intellectual disabilities, autism, epileptic encephalopathies, and other congenital disorders.

Since only 5 to 10 cells can be biopsied from an IVF blastocyst embryo, the DNA is amplified before being sequenced and read for diagnosis. However, the amplification process itself can introduce thousands of errors which appear to be de novo mutations. Until now, it has been difficult to distinguish sequencing errors from true de novo mutations. However, using their previously published method of Long Fragment Read (LFR) technology, Yale researchers assigned DNA fragments to either maternal or paternal genomes using DNA barcodes.

With the LFR technique, the scientists were able to remove over 100,000 sequencing errors, reducing the error rate 100 fold over previous studies.

"Because individuals carry on average less than 100 de novo mutations, being able to detect and assign the parent of origin for a mutation — and the cause of disease — requires an extremely low error rate."

Brock A. Peters PhD and Radoje Drmanac PhD, both of Complete Genomics, Inc., Mountain View, California, and co-corresponding authors.

Overall, the researchers detected 82% of all de novo changes in the IVF embryos. This is the first demonstration that a large majority of single base de novo mutations could be detected in a Preimplantation Genetic Diagnosis (PGD) test.

Adding to the complexity of this new technology is understanding each gene product. In one embryo, researchers did not find any de novo mutations in protein-coding regions of the genome. However, in another embryo from the same couple, researchers found two coding mutations in the ZNF266 and SLC26A10 genes that may be potentially harmful. But, the authors point out that it is currently unknown if there would be any health consequences for a child born with these particular gene mutations.

"The biggest hurdle now is how to analyze the medical impact of mutations detected — and make decisions based on those results."

Brock A. Peters PhD and Radoje Drmanac PhD.

In addition to PGD, this new technology could be useful in other applications where cells to be diagnosed are limited, such as circulating tumor cells (CTCs) or circulating fetal cells (CFCs), each of which are in rare subpopulations which exist in the blood supply.

Abstract
Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ~100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.

Authors: Peters BA, Kermani BG, Alferov O, Agarwal MR, McElwain MA, Gulbahce N, Hayden DM Tang YT, Zhang RY, Terle R, Crain B, Prates R, Berkeley A, Munné S, Drmanac R. 2015. Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing. Genome Res doi: 10.1101/gr.181255.114

About Genome Research:
Launched in 1995, Genome Research is an international, continuously published, peer-reviewed journal that focuses on research that provides novel insights into the genome biology of all organisms, including advances in genomic medicine. Among the topics considered by the journal are genome structure and function, comparative genomics, molecular evolution, genome-scale quantitative and population genetics, proteomics, epigenomics, and systems biology. The journal also features exciting gene discoveries and reports of cutting-edge computational biology and high-throughput methodologies.

About Cold Spring Harbor Laboratory Press:
Cold Spring Harbor Laboratory Press is an internationally renowned publisher of books, journals, and electronic media, located on Long Island, New York. Since 1933, it has furthered the advance and spread of scientific knowledge in all areas of genetics and molecular biology, including cancer biology, plant science, bioinformatics, and neurobiology. The Press is a division of Cold Spring Harbor Laboratory, an innovator in life science research and the education of scientists, students, and the public. For more information, visit our website at http://cshlpress.org/

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